CD44 Mediated Nonviral Gene Delivery into Human Embryonic ...publish.illinois.edu/cheng-research-group/files/2017/01/2016-Yen... · CD44 Mediated Nonviral Gene Delivery into Human

CD44 Mediated Nonviral Gene Delivery into Human Embryonic StemCells via Hyaluronic-Acid-Coated NanoparticlesJonathan Yen,†,§ Hanze Ying,‡ Hua Wang,‡ Lichen Yin,⊥ Fatih Uckun,||,¶ and Jianjun Cheng*,‡,†

†Department of Bioengineering and ‡Department of Materials Science and Engineering, University of Illinois at Urbana−Champaign,Urbana, Illinois 61801, United States⊥Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials,Collaborative Innovation Center of Suzhou, Nano Science and Technology, Soochow University, Suzhou 215123, PROC||Developmental Therapeutics Program, Children’s Hospital Los Angeles, Children’s Center for Cancer and Blood Diseases, LosAngeles, California 90027, United States¶Department of Pediatrics and Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine,Los Angeles, California 90027, United States

ABSTRACT: Gene delivery is an important tool to study and manipulatehuman pluripotent stem cells for regenerative medicine purposes. Yet currentmethods of transient gene delivery to stem cells are still inefficient. Throughthe combination of biologically based concepts and material design, we aim todevelop new methods to enhance the efficiency of gene delivery to stem cells.Specifically, we use poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-L-glutamate) (PVBLG-8), a membrane-active helical, cationic polypeptide, tocondense plasmid DNA to form stable nanocomplexes, which are furthercoated with hyaluronic acid (HA). HA not only shields the positive charges ofPVBLG-8 to reduce toxicity, but also acts as a targeting moiety for cell surfacereceptor CD44, which binds HA and facilitates the internalization of thenanocomplexes. Upon entering cells, HA is degraded by hyaluronidase inendosomes and PVBLG-8 is exposed, facilitating the endosomal escape ofDNA/polypeptide complex. Our studies show that the coating of HAsignificantly increases gene transfection efficiency of DNA/PVBLG-8 nanocomplexes from about 28 to 36% with largely reducedtoxicity.

KEYWORDS: human embryonic stem cells, nonviral gene delivery, polypeptides, human pluripotent stem cells, hyaluronic acid, CD44

1. INTRODUCTION

Human embryonic stem cells (hESCs) hold tremendouspotential in the field of regeneration due in particular to twocharacteristics: pluripotency and self-renewal. In pursuit ofregenerative medicine, gene delivery is an important tool tomanipulate and control cell fate. The control and over-expression of specific genes afforded by gene delivery isvaluable not only in efforts to control stem cell fate1−4 and forgene targeting studies.5,6 Currently, viral gene delivery is stillthe tool of choice for gene overexpression in hESCs formechanistic and differentiation studies, due to its hightransfection efficiency and stable transgene expression.7 Butviral gene delivery is plagued with issues that are unsuitable forbiomedical applications, including random viral integration intothe host, high cytotoxicity, immunogenicity, and insertionalmutagenesis.8,9 Nonviral gene delivery has gradually become animportant tool for stem cell engineering to transiently expressgenes of interest with reduced toxicity, providing a promisingalternative to viral gene delivery.10−16

There have been various materials developed for nonviralgene delivery into a variety of cells such as poly beta amino

esters17 and helical polypeptides.18,19 However, many issuesremain to be addressed in nonviral gene delivery, particularly inhESCs, such as low transfection efficiency and high cytotoxicity.Because of the physiology of hESCs, nonviral gene delivery tohESCs usually has low uptake efficiency.17,20 We have reportedthat commonly used nonviral gene delivery materials includingPEGylated PVBLG-8, polylysine (PLL), polyethylenimine, andcommercially available Lipofectamine 2000 demonstratestransduction efficiencies of 8, 3, 8, and 10% in hESCs,respectively.18,20 Green group’s poly beta amino estersdemonstrated transfection efficiency of 15−20%.17 Effortshave been made to improve the cell uptake of nanoparticlesby modifying their surface with certain targeting ligands thatcan specifically bind to cell surface protein receptors.21−26 Afterentering cells, the DNA/plasmids need to reach the nucleus fortranscription, which instead are usually trapped in theendosomes and thus tagged for degradation. We thus aimed

Received: September 11, 2015Accepted: January 26, 2016Published: February 23, 2016

Article

pubs.acs.org/journal/abseba

© 2016 American Chemical Society 326 DOI: 10.1021/acsbiomaterials.5b00393ACS Biomater. Sci. Eng. 2016, 2, 326−335

pubs.acs.org/journal/abseba

http://dx.doi.org/10.1021/acsbiomaterials.5b00393

to develop an effective delivery system with active targetingmotif coated on the complex, which not only can enhance theuptake of the gene delivery vehicles and shield the cationicpolymers to reduce cytotoxicity, but can also quickly escapefrom endosome after rapid dissociation of the charged shieldingmoiety in the endosome.Poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-L-

glutamate) (PVBLG-8), a novel helical, charged polypeptidedeveloped in our group, has been demonstrated to possessexcellent membrane disruption and endosomal escape capa-bility as a gene delivery system,27−42 which can be attributed tothe cationic charge and rigid α-helical structure.19,37,43 AlthoughPVBLG-8 shows some gene transfection efficiency in hESCs, itexhibits typical charge-associated cytotoxicity to the cells.19 Ourlab has developed multiple methods to modify the cationichelical polypeptide to reduce its toxicity, such as structuralreconfiguration,29 conjugations,18,28,31 and incorporation oftrigger-degradable protection group,32 while retaining orincreasing its transfection efficiency through enhanced endo-somal escape capability. Herein, we report a new gene deliverysystem based on self-assembled DNA/PVBLG-8 nanocom-plexes coated with negatively charged hyaluronic acid (HA).HA can specifically bind to CD44, a known cell surface receptorwith high expression level in hESCs.44 By coating the DNA/PVBLG-8 nanocomplexes with negatively charged HA, it notonly shields the positive charges of PVBLG-8 to decrease thetoxicity of nanocomplexes, but also acts as a targeting moietyfor receptor based endocytosis through HA/CD44 interactions

in hESCs, enhancing cellular uptake. Furthermore, the HA canbe deshielded by endogenous hyaluronidase in endosomes aftercellular internalization to expose the membrane-active cationichelical PVBLG-8, allowing rapid and efficient endosomalrelease and expression of DNA plasmids (Scheme 1). Weexpect the new design will lead to higher hESC transfectionefficiency and lower cytotoxicity.

2. MATERIALS AND METHODS2.1. General. hESCs H1 (hESC-H1) was cultured in E8 medium

from Stem Cell Technologies (Vancouver, Canada). Humanmesenchymal stem cells (hMSCs) were purchased form Lonza(Basel, Switzerland). COS-7 cells were purchased from ATCC(Manassas, VA, USA). Y-27632 was purchased from Stemgent(Cambridge, MA, USA). YOYO-1 was purchased from Invitrogen(Carlsbad, CA, USA). pEGFP-N1 was obtained from ElimBiopharmaceuticals (Hayward, CA, USA). Milli-Mark Anti-SSEA-4-PE was purchased from EMD Millipore (Billerica, MA, USA).PVBLG-8, a helical cationic polypeptide with a degree of polymer-ization of 200, was synthesized following previously reportedprocedures.45

2.2. Instrumentation. Flow cytometry analysis was conducted ona BD LSRII flow cytometry analyzer (Becton Dickinson, FranklinLakes, NJ). Cells were visualized with a Zeiss Axiovert 40 CFLfluorescence microscope equipped with 10 and 20x objectives(Thornwood, NY). Zeta potential and size analysis was conductedon the Malvern Zetasizer (Worcestershire, UK).

2.3. Nanocomplex Formation and Characterization. Variousnonviral vectors were used for the transfection analyses. Plasmid DNA(1 μL, 1 mg/mL) was diluted in water (25 μL). PVBLG-8 (7.5 μL, 1

Scheme 1. Formulation and Uptake Route of the Hyaluronic-Acid-Coated PVBLG-8/DNA Nanocomplexes; (1) HA-CoatedNanocomplexes Target and Bind to CD44 Receptors; (2) Nanocomplexes are Uptaken through Receptor-MediatedEndocytosis; (3) Low pH and Hyaluronidase Break down Nanocomplexes within Endosomes; (4) PVBLG-8 Peptides Breakthrough Endosome Membranes and Allow Cargo to Escape

ACS Biomaterials Science & Engineering Article

DOI: 10.1021/acsbiomaterials.5b00393ACS Biomater. Sci. Eng. 2016, 2, 326−335

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mg/mL) was diluted with water (25 μL). For the PVBLG-8 andcoated nanocomplex procedures, the PVBLG-8 and plasmids weremixed at various ratios and were incubated for 20 min at rt for complexformation. Hyaluronic acid (2.5−50 μL, 1 mg/mL) in water was addedto the nanocomplex mixture and incubated for another 20 min at rt.For the mixed system, the DNA and hyaluronic acid were first mixedtogether at various ratios and then PVBLG-8 was added. The mixturewas incubated at rt for 30 min for complex formation. Dynamic lightscattering (DLS) and zeta potential analysis were conducted on thesamples with a Malvern Zetasizer (Worcestershire, UK). Thenanocomplexes were subjected to electrophoresis in 1% agarose gelat 100 V for 45 min to evaluate DNA condensation by thepolypeptides in terms of DNA migration.2.4. In Vitro Gene Transfection. Cells were seeded in matrigel

coated 24 well plates as single cells using accutase and incubated for 24h with rho-kinase inhibitor Y-27632 (10 μM). Using fresh low proteinand serum free E8 medium, nanocomplexes as described earlier wereadded at 1 μg DNA/well. After incubation for 4 h, the medium wasreplaced with fresh E8 medium and cells were cultured for 48 h. Thecells were collected with accutase, fixed with paraformaldehyde andanalyzed using flow cytometry to determine transfection efficiency.2.5. In Vitro HA Blocking. hESCs were incubated with free

hyaluronic acid (300 kDa, 1−5 mg/mL) for 1 h prior to transfection.The cells were then washed 1x with PBS and placed back with fresh E8media. The nanocomplexes were added to the cells at 1 μg DNA/well.After incubation for 4 h, the medium was replaced with fresh E8medium and cells were cultured for 48 h. The cells were collected withaccutase, fixed with paraformaldehyde and analyzed using flowcytometry to determine transfection efficiency.2.6. pH and Hyaluronidase Exposure of Nanocomplex.

Nanocomplexes were formulated as previous described. HA coatednanocomplexes (400 μL, 100 μg/mL DNA) were incubated withhyaluronidase (100 μL, 0.5 mg/mL) in phosphate and citric acid bufferwith a pH of 7.4, 6.8, 6.2, or 5.6 for 0, 30, or 120 min at 37 °C. TheHA coated nanocomplexes (100 μL) were then diluted in theircorresponding buffer and the zeta potential of the resulting particleswas analyzed with the Zetasizer.To test the membrane activity of the nanocomplexes, we formulated

large unilamellar vesicles by extrusion technique (LUVETS)46 loadedwith 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX).47 Briefly, DOPC (100 μL, 25 mg/mLCHCl3) and POPC (1 mL, 10 mg/mL CHCl3) were mixed in round-bottom flask and evaporated with a rotary evaporator to create dry thin

film. The film was allowed to dry overnight under vacuum. The lipidfilm was rehydrated by 5 mL of ANTS/DPX solution (12.5 mMANTS, 45 mM DPX, 10 mM phosphate buffer, pH 7.4) for 30 min atrt. The solution was then put through 5 freeze thaw cycles in liquidnitrogen and lukewarm water. The solution was then extruded 15times through 0.4 μM polycarbonate membranes. Any extravesicularcomponents were removed through a Sephadex G-50 gel filtrationcolumn.

Nanocomplexes were formulated as previous described. HA coatednanocomplexes (400 μL, 100 μg/mL DNA) were incubated withhyaluronidase (100 μL, 0.5 mg/mL) in phosphate and citric acid bufferat pH 7.4 or 6.8 for 0 or 30 m at 37 °C. The HA coatednanocomplexes (10 μL) and the ANTS/DPX LUVETs (5 μL) weremixed and diluted by the corresponding buffer to a final solution of100 μL. Triton x was used as the positive control. The leaked ANTSdye was measured using a plate reader at 360 nm excitation and 530nm emission. The final leakage percentage was calculated using theratio of fluorescence of the sample with the positive control afterbackground subtraction.

2.7. Cell Viability. Cells were seeded in matrigel coated 96 wellplates as single cells using accutase and incubated for 24 h with y-27632 (10 μM). Using fresh low protein and serum free E8 medium,nanocomplexes were added at 0.2 μg DNA/well. After incubation for 4h, the medium was replaced with fresh E8 medium and cells werecultured for 48 h. Cell viability was evaluated through a MTT assay.Cells without complex treatment served as the control and results wereexpressed as percentage viability of control cells.

2.8. Intracellular Uptake Studies. DNA (1 mg/mL) was mixedand labeled with YOYO-1 (20 μM) at one dye molecule per 50 bp ofDNA. The HA coated nanocomplexes were then formed with YOYO-1/DNA as discussed before at a DNA:PVBLG-8:HA weight ratio of1:7.5:7.5. hESCs were plated on matrigel coated 24-well plate andallowed to grow to medium sized colonies. The complexes were addedto the wells at 1 μg of YOYO-1-DNA per well and incubated for 4 h at37 °C. The cells were then quantified through flow cytometry toquantify YOYO-1 uptake. Results are expressed as mean fluorescencelevels.

To elucidate the mechanisms regarding the cellular internalizationof DNA/PVBLG-8/HA complexes, we performed the uptake study at4 °C or in the presence of various endocytic inhibitors for 2 h. Cellswere pretreated with chlorpromazine (10 μg/mL), genistein (200 μg/mL), dynasore (80 μM), and wortmannin (50 nm) for 30 min beforethe complexes were added and throughout the experiment for 2 h at

Figure 1. Characterization of HA-coated nanocomplexes at 1:7.5 DNA:PVBLG-8 weight ratios (a) Gel retardation of varying HA-coatednanocomplexes. (b) Gel retardation of a HA mixed with DNA nanocomplexes at 1:7.5:7.5. (c) Size and zeta potential of HA coated nanocomplexes.(d) Stability of HA coated nanocomplexes (1:7.5:7.5) in water, PBS, and E8 medium as determined by size change measured by DLS.



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37 °C. Results are expressed as percentage of the mean GFPfluorescence level at 37 °C in control cells in the absence of endocyticinhibitors.2.9. Western Blot Analysis and SSEA Staining. After 72 h of

transfection, the cells were stained with DAPI (250 μL, 3 nM) andSSEA-4−PE (250 μL, 0.02 mg/mL), a pluripotency cell marker, for 30min at 37 °C. After 5 d, the cells were collected with RIPA buffer andmixed with Laemmli buffer supplemented with 2-mercaptoethanol,heated at 100 °C for 5 min to denature the proteins and then put onice. The samples were ran on 10% SDS PAGE Gel at 120 V for 1.5 h,and wet transferred to the nitrocellulose membrane using theAMRESCO Rapid Western Blot Kit per manufacturer’s instructions.The membrane was stained with OCT4 and α-Tubulin primaryantibodies and then with HRP-tagged secondaries.2.10. Statistical Analysis. All statistics analysis were performed by

GraphPad Prism v6.07. Multiple group comparisons were analyzedusing one-way ANOVA analysis with Dunnet’s post-testing with1:7.5:0 as control, n = 3, and with significance of p < 0.05. Significancebetween two groups were performed by student t test, n = 3, and withsignificance of p < 0.05 and p < 0.01.

3. RESULTS3.1. Characterization of DNA:PVBLG-8:HA Nanocom-

plexes. DNA condensation by PVBLG-8 followed by HAcoating, through charge interaction of the negatively chargedHA and the positively charged PVBLG-8, was evaluated by gelretardation assay. As shown in Figure 1a, DNA can be boundtightly to PVBLG-8 with a weight feeding ratio of 1:7.5.Coating of HA on the nanocomplex did not disrupt or displacethe DNA in the nanocomplexes at the feeding ratio of DNA toPVBLG-8 to HA ranging from 1:7.5:0 to 1:7.5:50. These resultsindicate that the complexes are stable through the coating, andthe negative charges on the HA does not interfere in theassembly stability and does not lead to disassembly of thenanocomplexes. Yet, when the HA was mixed directly with theDNA and PVBLG-8, the complex was unstable and some DNAleakage was detected (Figure 1b), substantiating the importance

of preforming DNA/PVBLG-8 complex prior to the coatingwith HA.The size and zeta potential of the DNA/PVBLG-8

nanocomplexes at 1:7.5 weight ratios with varying HA weightratio coatings were characterized using the DLS and zetasizer(Figure 1c). Without HA coating, the nanocomplex size was57.9 nm in diameter. After the coating of HA, at between1:7.5:5 and 1:7.5:15 weight ratios, the effective diametersincreased to and stabilized around 170 nm. With furtheraddition of HA to 1:7.5:50, the diameter increased to 270 nmwith an additional peak at 5.1 μm (data not shown), suggestingsubstantial change of the morphology and aggregation of thenanocomplex and presumably weakened interaction of DNA/PVBLG-8, although leakage of DNA was not observed. Thisobservation also clearly showed the dynamic interaction of HAwith the nanocomplex. The zeta potential of the nanocomplexwithout HA, measured to be 19.2 mV, decreased to 11.1 mVwith HA coating at 1:7.5:1 ratio, indicating that thenanocomplexes retained their positive charges with surfacepartially covered by HA. The zeta potential then decreases tobetween −18 and −25 mV when the ratio of DNA:PVBLG-8:HA was between 1:7.5:5 and 1:7.5:15, and to −42 mv atDNA:PVBLG-8:HA ratio of 1:7.5:50. The increase of the sizeand decrease of zeta potential suggest that formation ofnanocomplexes with HA coating can be achieved via a dynamicprocess and the amount of HA on nanocomplex surface can bemodulated by the amount HA added, but materials and chargebalance are critical to the intended functions nanocomplexesand should impact their gene delivery efficiency to cells. HAwith transient stability on the nanocomplex surface enhancesuptake, contributes to the desired dissociation of HA from thenanocomplexes upon internalization into the endosomes, andexposes PVBLG-8 for endosomal release.The stability of the nanocomplexes at the optimal

DNA:PVBLG-8:HA ratio (1:7.5:7.5) was selected due to the

Figure 2. HA-coated nanocomplexes significantly enhance the transfection efficiency of GFP encoding plasmid in hESCs. (a) Flow cytometryanalysis of transfection efficiency of hESCs with varying ratios of HA coating on DNA:PVBLG-8 nanocomplexes. (One-way ANOVA analysis withDunnet’s post-testing with 1:7.5:0 as control, n = 3 * p < 0.05 error bars: Std. Dev.) (b) Fluorescence images of H1 hESCs 48 h post transfectionwith and without HA coated nanocomplexes and with 5 mg/mL free HA pretreatment (scale bar =250 μm). c) Flow cytometry analysis oftransfection comparison between CD44(+) hMSCs and hESCs, and CD44(−) COS-7 cells to demonstrate the effect of HA targeting. (t testcomparison n = 3 * p < 0.05 ** p < 0.01, error bars: Std. Dev.) d) Transfection of 1:7.5:7.5 nanocomplexes in cells pretreated with 0, 2.5, or 5 mg/mL of free HA. (t test comparison n = 3 * p < 0.05 ** p < 0.01, error bars: std. dev.).



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highest transfection efficiency in hESCs (see Figure 2a) andtested in water, PBS and E8 medium through the use of thedynamic light scattering to determine the size of thenanocomplexes over time under various conditions (Figure1d). The particle stability was tested for up to 4 h (typicalincubation time for transfection) after formulation at 37 °C. Weassumed that if the HA coating was unstable, the size of thenanocomplex would likely increase due to charge-inducedaggregation. Experimental results showed that the sizes ofnanocomplex remained constant in different media over time,which indicated that the nanocomplexes are quite stable.3.2. hESC Transfection with HA-Coated DNA/PVBLG-8

Nanocomplexes. Through the optimization of variousDNA:PVBLG-8 weight ratios, 1:7.5 was found to be theoptimal ratio that gave the highest transfection efficiency (datanot shown). Thus, the DNA:PVBLG-8 ratio was fixed at 1:7.5with various ratios of HA coating on the nanocomplexes.Without HA coating, the nanocomplexes achieved 22%transfection efficiency, while with the increasing ratio of HAcoated on the nanocomplexes, the transfection efficiency wentup to 28%, 27% and 30% for 7.5:2.5, 7.5:5, 7.5:7.5 of PVBLG-8:HA ratios, respectively. As we increased the amount of HA to15 and 30, the transfection efficiency decreased to 20 and 17%,respectively (Figure 2a, b). The decrease in transfectionefficiency might be attributed to the excess HA in the systemthat does not coat the nanocomplexes but competes for thebinding with CD44 receptors. PVBLG-8 by itself showedmedium toxic to the cells (Figure 3a). With HA coating,nanocomplexes showed reduced toxicity and higher trans-fection.To demonstrate that the targeting of the HA coated

nanocomplexes were due to the CD44 binding of the HAcoated on the surface, pretreatment of free HA was applied tothe cells for 30 min and then washed off before transfection.With the pretreatment of 2.5 and 5 mg/mL of free HA, a dropof transfections to 8 and 6% were shown, respectively (Figure2d). This indicates that free HA bound to the cell surface CD44receptors block the uptake of nanocomplex, suggesting CD44-mediated uptake of the nanocomplex. To further demonstratethe importance of the CD44 receptor for the system, the HAcoated nanocomplexes were transfected in CD44 positivehuman mesenchymal stem cells (hMSCs) and CD44 negativeCOS-7 cells (Figure 2c). For CD44 positive hMSCs, HAcoated nanocomplexes showed almost twice the transfectionefficiency than that without HA coating. In contrast, in CD44negative COS-7 cells, the transfection efficiency was 36%without the HA coating and it dropped down to 15% after HAcoating (Figure 2d), which was probably due to the negativecharge of HA shielding the nanocomplexes. These trendsdemonstrates the important roles the CD44 receptors play inthe transfection, which may be due to the increased uptake ofthe HA-coated nanocomplexes.3.3. Toxicity and Maintenance of hESCs. Cytotoxicity of

the HA coated complexes was evaluated in hESCs 48 h aftertransfection using the MTT assay. PVBLG-8 demonstrated acell viability of 75% (Figure 3a). But with the HA coatednanocomplexes, the cell viability increased to approximately90% with the HA coated at 1:7.5:7.5 weight ratios (Figure 3a).To ensure that the materials did not alter the cell’s behavior orcause any differentiation, we stained the cells with SSEA-4antibodies, stage specific embryonic antigen-4, 72 h aftertransfection, in which the cells all stained positive (Figure 3b).In addition, cell lysate were collected 5 days after transfection

and a Western blot was conducted to confirm the continuedexpression of the pluripotent factor, OCT4 (Figure 3c). Theexpression of SSEA-4 and OCT4 demonstrated that this systemdoes not cause undesired differentiation or interferes with thepluripotency of the hESCs.

3.4. Mechanistic Studies of DNA/PVBLG-8/HA Nano-complexes. To demonstrate that the enhanced gene trans-fection is due to the increased uptake through the CD44receptor mediated pathway, the total uptake of DNA wasevaluated. Using DNA tagged with YOYO-1, the amount ofDNA/YOYO-1 uptake into the cells was determined. FromFigure 4, it can be seen that without HA coating, the meanYOYO-1 fluorescence level measured to be about 61%, whereaswith the HA coating at 1:7.5:7.5 ratio, the mean YOYO-1fluorescence increased to 67%. In addition, with the treatmentof free HA, the mean YOYO-1 fluorescence level declinesignificantly to around 44%, indicating a reduced nanocomplexuptake. Alternatively, the uptake of DNA/YOYO-1 in CD44negative COS-7 cells is reduced significantly after the coating ofHA, further demonstrating that the CD44 receptor mediatedthe uptake of HA coated nanocomplexes (Figure 4c).To demonstrate that the HA coated nanocomplexes were

indeed targeting the CD44 receptors, hESCs were stained withCD44 antibodies and incubated with HA-coated DNA/YOYO-1/PVBLG-8 nanocomplexes for 2 h at 4 °C. Through confocal

Figure 3. HA-coated nanocomplexes do not compromise pluripotencyand reduces cell toxicity. (a) Cell viability of hESCs at varying ratios ofHA coating of nanocomplexes as determined by MTT assay. (One-way ANOVA analysis with Dunnet’s post-testing with 1:7.5:0 ascontrol, n = 3, * p < 0.05) b) DAPI and SSEA4 staining patterns ofhESCs 72 h after transfected with HA coated nanocomplexes (scalebar = 250 μm). (c) Western blot analysis shows OCT4 expression inhESCs 5 days post HA-coated nanocomplexes transfection, thebottom two bands are nonspecific.



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imaging, CD44 is stained uniformly across the surface of thehESCs. The DNA/YOYO-1 can be visualized on the cellsurface colocalized with the CD44 receptors, indicating theimportance of the roles of the CD44/HA interactions (Figure4b).To elucidate the mechanisms underlying the cellular

internalization of the HA-coated nanocomplexes, we performedthe uptake study at 4 °C or in the presence of various endocyticinhibitors. Energy-dependent endocytosis was blocked at 4 °C;clathrin-mediated endocytosis was blocked by chlorpromazine,which triggers dissociation of the clathrin lattice; caveolae wasinhibited by genistein and mβCD by inhibiting tyrosine kinaseand depleting cholesterol, respectively; macropinocytosis wasinhibited by wortmannin by inhibiting phosphatidyl inositol-3-phosphate.48,49 When the cells were incubated at 4 °C, theuptake dropped to almost 3% of the control, whereaschlorpromazine dropped to 62%, genistein dropped to 22%,and wortmannin to 91% of the control (Figure 5a). Thedramatic decrease of transfection efficiency at 4 °C indicates the

process is energy dependent, and with the decrease of uptakewith the treatment of chlorpromazine and genistein, furtherdemonstrates that the nanocomplexes enter the cells throughreceptor mediated endocytosis.Because of the high membrane disruption ability of PVBLG-

8, the YOYO-1-DNA can quickly escape the endosomes andpermeate into the cell cytoplasm after 2 h incubation at 37 °C(Figure 5b). PLL, a substitute cationic peptide with lowermembrane disruption ability was used to visualize the uptakeand localization of the HA coated complexes. Through confocalimaging, there is significant colocalization of the HA coatedYOYO-1-DNA/PLL complex with the Lysotracker red stain asseen through CLSM confocal imaging (Figure 5b). Itdemonstrates that HA coating facilitates the nanocomplexuptake through receptor mediated endosomal pathway. But italso showed that PVBLG-8 nanocomplex induces moreefficient endosomal escape, which is beneficial for genetransfection.19 We demonstrated that PVBLG-8 is exposedfrom the nanocomplex through HA degradation to allow for

Figure 4. HA-coated nanocomplexes promotes cellular internalization of the gene cargo through CD44 receptor interactions. (a) Cell uptake level ofPVBLG-8/YOYO-1-DNA complexes in hESCs with varying amounts of HA coating and with the pretreatment of hESCs with free HA (n = 3). (b)CLSM images of hESCs incubated with HA coated PVBLG-8/YOYO-1-DNA complexes at 4 °C for 1 h with CD44-PE antibody staining (scale bar= 10 μm). (c) Cell uptake level of YOYO-1-DNA/PVBLG-8 complexes with and without HA coating in CD44 negative COS-7 cells at 1:7.5 weightratios. (t test comparison n = 3 * p < 0.05 ** p < 0.01, error bars: std. dev.).



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membrane disruption and endosomal escape once the nano-complex reaches the endosomes in the following studies.3.5. Exposure of PVBLG-8 from HA-Coated Nano-

complex for Endosomal Escape. The rapid endosomalescape of the nanocomplex was presumably due to PVBLG-8exposure as the HA coating is released and digested in theendosomal environment. To investigate the mechanism ofendosome triggered membrane disruption of HA-coatednanocomplexes for rapid endosomal escape, we evaluated thenanocomplex zeta potentials and membrane disruption abilitiesafter treatment with hyaluronidase (HAase) at varyingendosomal pH. At the initial time point of incubation of theHAase with varying pH of 7.4, 6.8, 6.2 and 5.6, the zetapotential remains negative at −21, −17.6, −16.8, and −10.3mV, respectively. After 30 min incubation, the zeta potential atpH of 7.4 and 6.8 increased but remained negative at −18.2 and−8.32 mV, respectively. On the other hand, the zeta potentialat pH of 6.2, and 5.6, all increased to the positive range of 1.34and 9.39 mV, respectively. Finally, after 120 min the zetapotential at pH of 7.4 stays negative at −9.3 mV. But, for thepH at 6.8, 6.2 and 5.6, the zeta potential increases to positive of1.69, 7.2, and 11.8 mV, respectively. A pH of 7.4 isrepresentative of the cytoplasmic pH, whereas early and lateendosomes have a pH of 6.8 and 5.6, respectively50 (Figure 6a).The shift of zeta potential toward positive indicates theexposure of the cationic helical peptides, which are then capableof membrane disruption.To further illustrate that the membrane disruption ability of

the HA-coated nanocomplexes at an early pH endosome of 6.8is due to the HAase degradation rather than pH-inducedcomplex dissociation, large unilamellar vesicles (LUV) were

synthesized for dye leakage assay. At a pH of 7.4 and 6.8, it canbe seen that the non-HA-coated nanocomplexes allowed for100% release of the dye, whereas with HA coating, the

Figure 5. HA-coated nanocomplexes promotes cellular internalization of the gene cargo through endocytosis. (a) Cell uptake mechanisms of HAcoated PVBLG-8/YOYO-1-DNA complexes in hESCs. (n = 3). (b) CLSM images of uptake and endosomal escape of HA-coated PVBLG-8/YOYO-1-DNA and PLL/YOYO-1-DNA complexes and with Lysotracker staining (scale bar = 10 μm) (t test comparison n = 3 ** p < 0.01, error bars: std.dev.).

Figure 6. pH and HAase effect on HA-coated nanocomplexes. (a)Changes in the zeta potential of DNA/PVBLG-8/HA nanocomplexesafter treatment with 0.5 mg/mL of HAase at different pH values fordifferent times. (b) ANTS/DPX-loaded LUVET leakage study ofdifferent nanocomplexes with and without the HAase treatment atdifferent pH values.



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nanocomplexes only demonstrated slight membrane disruptionat pH of 7.4, and a slight increase in membrane disruption at apH of 6.8. Alternatively, with the addition of HAase after 30min incubation at pH of 7.4 and 6.8, the dye leakage increasedup to 46% and 76%, respectively, indicating strong membranedisruption ability (Figure 6b). In addition, nanocomplexesusing PLL were also studied and it was found that with orwithout HA coating, there was no membrane disruption asexpected. These studies indicated that the HA coated PVBLG-8nanocomplex is responsive to the endosomal environment withenhanced HAase activity induced by low pH, allowing for rapidendosomal escape with the exposed PVBLG-8.

4. DISCUSSIONWe developed a novel gene transfection system that contains anouter negatively charged cell targeting shell consisting of HA,with an inner core made from a cationic helical peptide capableof condensing DNA and facilitating endosomal escape. Thesystem utilizes the charge interactions between the outernegatively charged shell and positive inner core to stabilize thenanocomplex at neutral pH, but allows for the deshelling andexposure of the positive helical peptides when exposed to alower pH and HAase within the endosome. With the HAcoating of the nanocomplexes, the hESC transfection efficiencyof DNA/PVBLG-8 nanocomplexes increases significantly,despite the alteration of the overall surface charge from positiveto negative. HA coated nanocomplexes also lower the toxicityof the cationic helical peptide and have little effect on the cellspluripotency, for hESCs were still both OCT4 and SSEA4positive after materials treatment. HA is a negatively chargedbiocompatible polysaccharide that is found naturally in thebody and has been used as the backbone of many hydrogelsystems for stem cell engineering.44 A few glycosaminoglycans,except for hyaluronic acid have been shown to disrupt cationicpolypeptide gene transfections.51 The reversal of chargeindicates the loss of the initial membrane disruption effect ofthe cationic helical peptide, which in theory should havedecreased transfection. However, this study showed that thenegative nanocomplexes increased the transfection efficiency,indicating that the HA on the nanocomplexes are an integralpart to the transfection efficiency, in which they target theCD44 receptors that are expressed on hESC surfaces. We foundthat at weight ratios of 1:7.5:0 to 1:7.5:7.5, the transfectionefficiency increases, which then decreases with additional HAcoating. This decrease is likely to be caused by the excess HAthat was not coated on the nanocomplex. This can be seen bythe DLS and zeta potential, that after a certain ratio, the sizeand zeta potential of the nanocomplex stays constant. Thus, theexcess free HA could be competitively binding with the CD44receptor against the nanocomplexes demonstrated by thedrastic drop in transfection efficiency with the pretreatment ofHA. The decrease in transfection efficiency demonstrated thatthe free HA has bound to the cell surface CD44 receptors andblocked them from further uptake, indicating that the CD44/HA interactions is an important aspect to the increased efficacyof the nanocomplex. The HA coated nanocomplexes alsodemonstrated a higher efficacy in CD44 positive hMSCs, butsubstantially reduced transfection efficiency in CD44 negativeCOS-7 cells, substantiating the importance of the CD44receptor for the uptake of the HA coated nanocomplexes.To understand the mechanism of uptake, we conducted

uptake studies of DNA tagged with YOYO-1 dye and foundthat the cells did significantly increase the uptake of the

nanocomplexes with the HA coating. In addition, through thestudy with various endocytosis inhibitors, we found that at 4 °Cthe uptake was drastically inhibited, indicating that the uptakeof the particles were highly energy dependent. In addition, thedecrease of the uptake due to the chlorpromazine, demon-strated that some of the particles were internalized throughclathrin-dependent endocytosis. On the other hand, genisteinalso had a large decrease in uptake of the YOYO-1/DNA, thusindicating that some of the uptake is due to clathrinindependent pathways like caveolae mediated uptake. Allthese evidence showed that the materials were internalizedthrough receptor mediated pathways.Commonly used transfection materials like PLL have the

ability to condense DNA for nonviral gene delivery, but PLLdemonstrates no endosomal escape.52,53 Our nanocomplexsystem transforms to exhibit membrane disruptive properties inendosomal environment with high HAase activity induced bylow pH. The exposure of PVBLG-8 in the system enhances theendosomal release of the nanocomplex and thus the DNA.When the materials were switched to PLL, a nonmembranedisrupting polymer, there was little endosomal escape, and thenanocomplexes were mainly retained in the endosomes. Inearly endosomes, at a low pH and some HAase activity, the HAcoated nanocomplexes demonstrated increased LUV dyeleakage due to membrane disruption. Our system is superiorto other materials17,18,20 because of our highly available andactive membrane disruption activity that is initially shielded toreduce cytotoxicity to the cells, but is revealed when it entersthe endosomes. Thus, through the use of a targeting moiety fora receptor on hESCs is a step in looking at nonviral genedelivery into hESCs differently, in which a cell’s specificmechanism is exploited to enhance transfection efficiency.

5. CONCLUSION

We reported a new targeting and triggerable gene deliverysystem for human embryonic stem cells based on the PVBLG-8, a cationic helical polypeptide with high membrane disruptionproperties. The DNA/PVBLG-8 nanocomplex by itself wasdemonstrated to be toxic to the cells because of the highmembrane disruption properties. Using HA as a coating for thenanocomplexes not only decreased the toxicity from thecationic helical peptides, but also allowed targeting of hESCsand deshelling of charge shielding moiety that increased theuptake and release of DNA, which in turn increased thetransfection efficiency. This provides a promising approach tomanipulate hESCs or pluripotent stem cells through transientgene delivery, overcoming major hurdles toward developmentof various biomedical applications. Increased gene transfectionefficiency reduces the need for enrichment and sorting of themanipulated pluripotent stem cells. These preliminary studiesdemonstrated an enhanced gene delivery system to undiffer-entiated hESCs through an alternate receptor-mediatedendocytosis pathway and allows for future modification withother targeting moieties on the PVBLG-8 based gene deliveryto target other receptors on other hard to transfect cells.

■ AUTHOR INFORMATION

Corresponding Author*E-mail: [email protected]. Tel: (217) 244-3924.



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mailto:[email protected]


Present Address§J.Y. is currently at NGx, Developmental and MolecularPathways, Novartis Institutes for BioMedical Research, Cam-bridge, Massachusetts, 02139, USA

Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work is supported by NIH (Director’s New InnovatorAward 1DP2OD007246 and 1R21EB013379 for J.C) for thebiological studies of the project and NSF (CHE 1153122) forthe synthesis and development of the polypeptide. J.Y. wasfunded at UIUC from National Science Foundation (NSF)Grant 0965918 IGERT: Training the Next Generation ofResearchers in Cellular and Molecular Mechanics andBioNanotechnology.

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